Method of making recombinant enzyme

ABSTRACT

The present invention provides a polypeptide selected from a polypeptide of subunit (A) or a polypeptide of subunit (B) of a prenyl diphosphate synthetase; a DNA coding for the polypeptide; a recombinant vector comprising the DNA; a transformant transformed with the vector; and a method for preparing an active type prenyl diphosphate synthetase. Also disclosed is a method for preparing an active type enzyme on which a specific property has been conferred, comprising mixing polypeptides of the two subunits of a heterodimeric enzyme, the polypeptides being derived from different organisms and one of the polypeptides having the specific property.

This application is a division of application Ser. No. 08/873,235 filedJun. 11, 1997 which is incorported herein in its entirety by referencethereto.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing the peptide of aprenyl diphosphate synthetase, a method for producing an active typeprenyl diphosphate synthetase, a DNA coding for the synthetase, arecombinant vector comprising the DNA and a transformant transformedwith the vector.

2. Description of the Prior Art

An extremely wide variety of isoprenoid compounds are found in naturalcreatures from bacteria to higher eukaryotes. For example, steroids,carotenoids, polyprenols which are sugar carriers, quinones, tRNAmodified with isopentenyladenine, prenylated proteins and the like maybe enumerated. All of these isoprenoids are biosynthesized throughprenyl diphosphate as an intermediate which is produced by a prenyldiphosphate synthetase (FIG. 1).

The "prenyl diphosphate synthetase" is a general term for those enzymeswhich catalyze a reaction that condensation-polymerizes prenyldiphosphate (an allylic primer) and 3-isopentenyl diphosphate (IPP) toproduce polyprenyl diphosphate.

Prenyl diphosphate synthetases are divided into two groups. One groupconsists of enzymes that catalyze a condensation reaction in which thedouble bond formed by each condensation of IPP is of E type. The othergroup consists of enzymes that catalyze a condensation reaction in whichthe double bond formed by each condensation of IPP is of Z type.Further, the maximum length of the isoprene chain which each prenyldiphosphate synthetase can produce is fixed. Since the hydrophobicproperty of a product varies depending of the isoprene chain length ofthe product, there is great difference in the mode of requirement forthe activity of enzymes. When bacterial enzymes are compared in terms ofthe mode of requirement, prenyl diphosphate synthetases are classifiedinto the following four groups.

(1) Prenyl diphosphate synthetase I (E type, short chain prenyldiphosphate synthetase)

(i) Geranyl diphosphate (GPP) synthetase (Sagami, H. et al., (1978)Biochem. Biophys. Res. Commun., 85, 575) (C₅ →C₁₀)

The expression "C₅ →C₁₀ " means that the subject synthetase catalyzesthe synthesis from a compound with 5 carbon atoms to a compound with 10carbon atoms (hereinafter, this indication has a similar meaning.)

(ii) Farnesyl diphosphate (FPP) synthetase (Takahashi, I. and Ogura, K.,(1981) J. Biochem. 89, 1581; Fujisaki, S. et al., (1986) J. Biochem.,99, 1327) (C₅ →C₁₅)

(iii) Geranylgeranyl diphosphate (GGPP) synthetase (Takahashi, I. andOgura, K., (1982) J. Biochem. 92, 1527; Sagami, H. and Ogura, K., (1981)J. Biochem., 89, 1573) (C₅ →C₂₀)

(2) Prenyl diphosphate synthetase II (E type, medium chain prenyldiphosphate synthetase)

(i) Hexaprenyl diphosphate (HexPP) synthetase (Fujii, H. et al., (1982)J. Biol. Chem., 257, 14610) (C₁₅ →C₃₀)

(ii) Heptaprenyl diphosphate (HepPP) synthetase (Takahashi, I. et al.,(1980) J. Biochem., 255, 4539) (C₁₅ →C₃₅)

(3) Prenyl diphosphate synthetase III (E type, long chain prenyldiphosphate synthetase)

(i) Octaprenyl diphosphate (OctPP) synthetase (Fujisaki, S. et al.,(1986), J. Biochem., 99, 1327) (C₁₅ →C₄₀)

(ii) Nonaprenyl diphosphate (NonPP) synthetase (Sagami, H. et al.,(1977) Biochemistry, 16, 4616) (C₁₀ →C₄₅) Decaprenyl diphosphate (DecPP)synthetase (Ishii, K. et al., (1983) Biochem. Biophys. Res. Commun.,116, 500) (C₁₅ →C₅₀)

(4) Prenyl diphosphate synthetase IV (Z type, long chain prenyldiphosphate synthetase)

(i) Z-nonaprenyl diphosphate synthetase (Ishii, K. et al., (1986)Biochem. J., 233, 773) (C₁₅ →C₄₅)

(ii) Undecaprenyl diphosphate (UPP) synthetase (Takahashi, I. and Ogura,K. (1982) J. Biochem., 92, 1527; Keenman, M. V. and Allen, C. M. (1974)Arch. Biochem. Biophys., 161, 375) (C₁₅ →C₅₅)

(iii) Dehydrodolichyl diphosphate (deDolPP) synthetase (Sagami, H. etal., (1989) Biochem. Biophys. Acta. 1002, 218) (C₁₅ →C₈₅₋₁₀₅)

Prenyl diphosphate synthetase I successively condensates 3-isopentenyldiphosphate (IPP) with dimethylallyl diphosphate (DMAPP) generated byisomerization of IPP as an allylic primer to thereby synthesize a shortchain, totally E type prenyl diphosphate with 20 or less carbon atoms.This product serves as a precursor for steroids, carotenoids orprenylated proteins. Further, geranyl diphosphate (GPP), farnesyldiphosphate (FPP) and geranylgeranyl diphosphate (GGPP) also serve as anallylic primer substrate for a medium- or long-chain prenyl diphosphatesynthetase.

Hexaprenyl diphosphate synthetase (HexPS) and heptaprenyl diphosphatesynthetase (HepPS) belong to prenyl diphosphate synthetase II. Theseenzymes synthesize hexaprenyl diphosphate and heptaprenyl diphosphate,respectively, without DMAPP nor GPP as a primer but using FPP as anallylic primer. The products are highly hydrophobic and serve asprecursors for the side chains of menaquinones or ubiquinones inorganisms having these enzymes. These prenylquinones play importantroles in the respiratory chain or the electron transport system inphotosynthesis.

Any member of prenyl diphosphate synthetase II is an enzyme composed oftwo essential proteins which do not have the catalytic activityindependently. However, the enzyme has a property that in the presenceof substrates for the enzyme, the two proteins associate with each otherand exhibit the catalytic activity (Yoshida, I. et al., (1989) Biochem.Biophys. Res. Commun., 160, 448). In this point, the enzyme of thisgroup is greatly different from other prenyl diphosphate synthetase.

As a microorganism producing prenyl diphosphate synthetase II,Micrococcus luteus, Bacillus subtilis and the like are known (Fujii, H.et al., (1982) J. Biol. Chem., 257, 14610; Takahashi, I. et al., (1980)J. Biol. Chem., 255, 4539). The following facts have been shown on thetwo components (designated "component A" and "component B") of HexPSfrom Micrococcus luteus B-P 26 and on the two components (designated"component I" and "component II") of HepPS of Bacillus subtilis.

a) Component A and component I are relatively high in thermostability,whereas component B and component II have thermostability as low as thatof other enzymes derived from mesophiles (Fujii, H. et al., (1982) J.Biol. Chem., 257, 14610).

b) There is no interchangeability between component A and component I.In other words, neither a combination of component A and component IInor a combination of component I and component B exhibits enzymeactivity (Fujii, H. et al., (1983) FEBS Lett., 161, 257).

c) Component B and component II are affected by SH reagent andarginine-specific reagent to lower the enzyme activity remarkably,whereas component A and component I are not affected by these reagents(Yoshida, I. et al., (1989) Biochem. Biophys. Acta, 995, 138).

Octaprenyl diphosphate synthetase (OctPS), nonaprenyl diphosphatesynthetase (NonPS) and the like belong to prenyl diphosphate synthetaseIII. Like prenyl diphosphate synthetase I, these enzymes are ahomodimeric protein composed of identical subunits. They exhibit thecatalytic activity by themselves. However, in order to maintain theturnover as a catalyst, they require a proteinaceous factor whichremoves hydrophobic products from their active site (Ohnuma, S. et al.,(1991) J. Biol. Chem., 266, 23706). This activator is interchangeableand exhibits activating action against any enzyme belonging to prenyldiphosphate synthetase III (Ohnuma, S. et al., (1991) J. Biol. Chem.,266, 23706).

Enzymes belonging to prenyl diphosphate synthetase IV condensate IPP inthe Z-structual form to synthesize polyprenyl diphosphate of E- and-Zmixed type using a short-chain prenyl diphosphate (GPP, FPP) as a primersubstrate. Bacterial undecaprenyl diphosphate synthetase (UPS) andeukaryotic dehydrodolichyl diphosphate synthetase (deDolPS) are includedin this group. A large number of these enzymes are a membrane-boundprotein and when solubilized with a surfactant or the like, they requirethe addition of a surfactant such as Triton X-100 for the manifestationof their activity in almost all cases (Takahashi, I and Ogura, K. (1982)J. Biochem., 92, 1527; Allen, C. M. and Muth, J. D. (1977) Biochemistry,16, 2908). Additionally, the activator common in prenyl diphosphatesynthetase III is ineffective against UPS. It is considered that thisfact is because hydrophobic environment of a membrane is essential forthe manifestation of the enzyme activity.

The most part of the above-described information has been obtained fromexperiments using those enzymes extracted and purified from a solutionof disrupted cells. In order to clarify a more detailed enzyme reactionmechanism, not only the primary structure but also the crystal structureof enzyme proteins should be analyzed. For this purpose, the cloning ofgenes coding for these enzyme proteins is indispensable.

Actually, prenyl diphosphate synthetase genes such as FPS and GGPS havebeen cloned recently one by one (FPP synthetases: Koyama, T. et al.,(1993) J. Biochem., 113, 355; Fujisaki, S. et al., (1990) J. Biochem.,108, 995; Anderson, M. A. et al., (1989) J. Biol. Chem., 264, 19176;Clarke, C. F. et al., (1987) Mol. Cell. Bio., 7, 3138; Wilkin, D. J. etal., (1990) J. Biol. Chem., 265, 4607; GGPP synthetases: Carattoli, A.et al., (1991) J. Biol. Chem., 266, 5854; Armstrong, G. A. et al.,(1990) Proc. Natl. Acad. Sci. USA, 87, 9975; Math, S. K. et al., (1992)Proc. Natl. Acad. Sci. USA, 89, 6761; Misawa, N. et al., (1990) J.Bacteriol., 172, 6704). With respect to HexPP synthetase, a gene codingfor one of the two components (corresponding to "component B" describedpreviously) has been cloned by an experiment on complementarity inyeast. However, the two components of this synthetase are necessary forthe manifestation of the activity, as described previously. Therefore,it cannot be said that a perfect cloning of the gene coding for theenzyme of active type has been performed (HexPP synthetase: Ashby, M. M.and Edwards, P. A. (1990) J. Biol. Chem., 265, 13157).

The present inventor has compared the primary structures of theabove-mentioned enzymes based on the base sequences for their genes. Asa result, it has become clear that prenyl diphosphate synthetases have 7regions in which the amino acid sequence has been relatively preservedbeyond the difference in chain length or organism species (Koyama, T. etal., (1993) J. Biochem. 113, 355-363). Since these regions are preservedin a group of enzymes which catalyze substantially the same reaction,they are believed to have an important role in the catalytic function.On the other hands, it is predicted that non-preserved regions have aportion defining the chain length, a portion involved in the differencein the mode of manifestation of the enzymatic function, and the like.However, at present, the number of cloned genes of prenyl diphosphatesynthetases having different chain length is too small to find out theexistence of such portions from comparison of primary structures.

From the viewpoint of the manifestation of enzymatic function, enzymesbelonging to prenyl diphosphate synthetase II are greatly different fromother prenyl diphosphate synthetases, as described previously. They arecharacterized by being composed of two proteins (heterodimeric type),each of which does not have a catalytic function alone but whichassociate with each other in the presence of a substrate to exhibit acatalytic function.

Substances synthesized by these heterodimeric prenyl diphosphatesynthetases are precursors of those substances such as vitamin K andubiquinones which exist universally in organisms and, thus, they areimportant physiologically active substances. Therefore, they are of highutility value. Furthermore, the prenyl diphosphate produced by aheterodimeric prenyl diphosphate synthetase is industrially extremelyuseful since the chain length and structural isomers thereof can bestrictly controlled. Thus, the expression of such a synthetase in largequantity is needed.

Accordingly, it is desired to isolate genes coding for the two proteinsof an enzyme belonging to prenyl diphosphate synthetase II, to expressthe genes separately and thereby to produce the proteins in largequantity.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for producingpeptides of prenyl diphosphate synthetases, a method for producing anactive type prenyl diphosphate synthetase, a DNA coding for thesynthetase, a recombinant vector comprising the DNA and a transformanttransformed with the vector.

As a result of extensive and intensive researches toward the solution ofthe above assignment, the present inventor has succeeded in cloning thegene of a prenyl diphosphate synthetase from Micrococcus luteus and alsosucceeded in preparing an active type prenyl diphosphate synthetase bymixing peptides of the individual subunits of a prenyl diphosphatesynthetase. Thus, the present invention has been achieved.

The present invention relates to a DNA coding for a polypeptide selectedfrom a polypeptide of subunit (A) of a prenyl diphosphate synthetase ora polypeptide of subunit (B) thereof.

Subunit (A) and Subunit (B) are two polypeptide chains which form aheterodimer to express prenyl diphosphate synthetase activity. Of thesechains, Subunit (B) has an amino acid sequence characteristic of prenyltransferase.

As the polypeptide of subunit (A), a polypeptide representedsubstantially by the amino acid sequence shown in SEQ ID NO: 1 may begiven. As the polypeptide of subunit (B), a polypeptide representedsubstantially by the amino acid sequence shown in SEQ ID NO: 2 may begiven. As the DNA coding for the polypeptide of subunit (A), the DNArepresented by SEQ ID NO: 3 may be given. As the DNA coding for thepolypeptide of subunit (B), the DNA represented by SEQ ID NO: 4 may begiven.

The term "substantially" used herein means that, as long as apolypeptide selected from a polypeptide of subunit (A) or a polypeptideof subunit (B) has activity to synthesize prenyl diphosphate, the aminoacid sequence for this polypeptide may have variations such as deletion,substitution, insertion, or the like.

Accordingly, for example, the amino acid sequence of SEQ ID NO: 1 havinga deletion of methionine (Met) at position 1 is included in theabove-mentioned amino acid sequence having variations. Also, not onlythe base sequence coding for the amino acids contained in thepolypeptide of the invention, but also a degeneracy isomer of the abovebase sequence different only in degenerate codons is included in the DNAof the present invention.

The present invention further relates to a recombinant vector comprisingthe DNA described above.

The present invention further relates to a transformant obtained bytransforming a host organism with the recombinant vector describedabove.

The present invention further relates to a method for producing apolypeptide of subunit (A) and/or a polypeptide of subunit (B)comprising culturing the transformant described above in a medium tothereby accumulate the polypeptide of subunit (A) and/or the polypeptideof subunit (B) in the culture and collecting the polypeptide(s).

The present invention further relates to a polypeptide selected from thepolypeptide of subunit (A) represented substantially by SEQ ID NO: 1 andthe polypeptide of subunit (B) represented substantially by SEQ ID NO:2.

The present invention further relates to a method for producing anactive type prenyl diphosphate synthetase comprising preparing peptidesof the individual subunits of a heterodimeric prenyl diphosphatesynthetase by recombinant DNA techniques and mixing the resultantpeptides of the individual subunits. As peptides of the individualsubunits, the polypeptide of subunit (A) and the polypeptide of subunit(B) represented by, for example, SEQ ID NO: 1 and SEQ ID NO: 2,respectively, may be given.

The present invention further relates to a method for producing anactive type enzyme comprising preparing separately polypeptides of thetwo subunits of a heterodimeric enzyme, the polypeptides being derivedfrom different organisms (e.g., microorganisms) and one of thepolypeptides having a specific property, and mixing the resultantpolypeptides for these subunits to thereby obtain an active enzyme onwhich the specific property has been conferred. In this method, as oneof the polypeptides for the two subunits, the polypeptide derived fromBacillus subtilis and represented substantially by the amino acidsequence of SEQ ID NO: 5 may be given. As the other polypeptide, thepolypeptide derived from Bacillus stearothermophilus and representedsubstantially by the amino acid sequence of SEQ ID NO: 6 may be given.As the active type enzyme on which the specific property has beenconferred, a thermoresistant, active type prenyl diphosphate synthetasemay be given.

The present invention further relates to a method for preparing anactive type prenyl diphosphate synthetase comprising mixing thepolypeptide of a subunit of a Bacillus subtilis-derived prenyldiphosphate synthetase represented substantially by the amino acidsequence of SEQ ID NO: 5 and the polypeptide of a subunit of a Bacillusstearothermophilus-derived thermoresistant prenyl diphosphate synthetaserepresented substantially by the amino acid sequence of SEQ ID NO: 6 tothereby obtain a heterodimeric, active type prenyl diphosphatesynthetase having thermal resistance and enhanced activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the biosynthetic pathway of isoprenoids.

FIG. 2 shows the designing of primers based on preserved amino acidsequences.

FIG. 3 shows a plate transfer device.

FIG. 4 presents autoradiograms showing the results of Southernhybridization.

FIG. 5 is a chromatogram showing the results of a reversed phase TLC.

FIG. 6 is a diagram showing a summary of deletion clones.

FIG. 7 is a diagram showing the ORFs of the DNA of the presentinvention.

FIG. 8 is a diagram showing a summary of those clones used in theconstruction of plasmids each having one of the ORFs.

FIG. 9 is a diagram showing plasmids each having one of the ORFs.

FIG. 10 presents the results of a reversed phase TLC (a chromatogram)showing the enzyme activity of the expression product when two plasmidseach having one of the ORFs are combined.

FIG. 11 presents the results of a reversed phase TLC (a chromatogram)showing the enzyme activity when the expression products of a Bacillussubtilis-derived gene and a Bacillus stearothermophilus-derived gene arecombined.

FIG. 12 is a graph showing the optimum temperature for each enzyme.

FIG. 13 is a graph showing the residual activity after heat treatment.

DETAILED DESCRIPTION OF THE INVENTION

Hereinbelow, the present invention will be described in detail.

Prenyl diphosphate synthetases have 7 regions in which the amino acidsequence has been relatively preserved beyond the difference in enzymesor organism species. It is expected that these regions are alsopreserved in the hexaprenyl diphosphate synthetase of Micrococcus luteusB-P 26 (obtained from Dr. L. Jeffries, Walton Oaks Experimental StationVitamins, Ltd.; hereinafter referred to as "M. luteus B-P 26").

Then, in the present invention, the prenyl diphosphate synthetase geneof M. Luteus B-P 26 is cloned using recombinant DNA techniques based onthe preserved amino acid sequences of bacterial prenyl diphosphatesynthetases.

Hereinbelow, techniques for DNA cloning will be described.

First, genomic DNA is prepared from a prenyl diphosphate synthetaseproducing bacterium, for example, cultured cells of M. luteus B-P 26.Subsequently, DNA probes are synthesized based on the 7 regions in whichthe amino acid sequence is relatively preserved beyond the difference inthe kind of prenyl diphosphate synthetase and microorganism species andcolony hybridization or the like is performed using these probes, tothereby clone a full length gene of interest.

(1) Preparation of Genomic DNA

The cultivation of M. luteus B-P 26 may be performed by conventionalmethods. For example, M. luteus B-P 26 is inoculated into a mediumcontaining 0.5% yeast extract, 1% polypeptide and 1% sodium chloride andcultured at 30-37° C. for 1-3 days. In order to prepare genomic DNA fromcultured cells of M. luteus B-P 26, any known technique may be used. Forexample, the cells are treated with lysozyme and then treated with asurfactant such as sodium lauryl sulfate. Thereafter, proteins areremoved from the cell lysate with an organic solvent such as phenol,chloroform, ether, or the like, and then the lysate are subjected toethanol precipitation. Thus, genomic DNA can be prepared easily byconventional methods (J. Mol. Biol., 3, 208, 1961).

Subsequently, the genomic DNA obtained is ligated to vector plasmids toprepare a genomic DNA library. This may be performed by conventionalmethods. For example, genomic DNA chains and plasmid DNA chains aredigested with an appropriate restriction enzyme (e.g., EcoRI, BamHI,HindIII, Sau3AI, MboI, PstI), and then the resultant DNA fragments aretreated with a DNA ligase (e.g., T4 DNA ligase) or, depending on thestate of the digested ends, with a terminal transferase or DNApolymerase. Thereafter, the DNA fragments are ligated using a DNA ligase(Molecular Cloning, Cold Spring Harbor Laboratory, 269, 1982; Method inEnzymol., 68, 41, 1979). As the vector used here, aλ phage vector (λgt10, Charon 4A, EMBL-3, etc.), a plasmid vector (pBR322, pSC101, pUC19,pUC119, pACYC117) and the like may be enumerated. The DNA fragmentsdescribed above are incorporated in these vectors, which are used totransform a host organism such as Escherichia coli (e.g., DH1, HB101,JM109, C600, MV1184, TH2) to thereby obtain a genomic DNA library.

(2) Preparation of Probes for Screening

First, probes for screening the above genomic DNA by hybridization areprepared. In order to prepare higher selective probes, it is consideredappropriate to prepare oligonucleotides coding for those regions inwhich amino acid residues are highly preserved among different speciesof organisms. Probes may be prepared by conventional chemical synthesis.As the amino acid sequence which satisfies the above condition, thefollowing preserved amino acid sequences are selected [the underlinedamino acids are preserved more than 50% in different species oforganisms (see below)] (FIG. 2).

The sequence "Gly Gly Lys Arg Ile Arg Pro Leu" (SEQ ID NO: 7) in RegionI

The sequence "Ser Leu Ile His Asp Asp" (SEQ ID NO: 8) and the sequence"Asp Leu Arg Arg Gly Arg Pro" (SEQ ID NO: 9) in Region II

The sequence "Leu Ala Gly Asp Gly Leu Leu" (SEQ ID NO: 10) in Region III

The sequence "Phe Gln Ile Arg Asp Asp Ile Leu Asp" (SEQ ID NO: 11) andthe sequence "Gly Lys Pro Val Gly Ser Asp" (SEQ ID NO: 12) in Region VI.

Regions I, II, III and VI used herein correspond to the regions ofpositions 39-52, positions 73-103, positions 115-123 and positions217-250, respectively, in the amino acid sequence for a Bacillusstearothermophylus-derived FPS described by Koyama, T. et al., J.Biochem. 113, 355-363 (1993).

The examination of preserved amino acid sequences among differentorganism species can be made among FPSs from Bacillusstearothermophylus, Escherichia coli, Saccharomyces cerevisiae, rat andhuman; GGPSs from Erwinia herbicola and Erwinia uredovora; and HexPSfrom Saccharomyces cerevisiae. The design of probes is performed basedon the amino acid sequences for the 7 regions of Bacillusstearothermophylus which belongs to Gram positive bacteria as M. luteusB-P 26 does.

Based on these amino acid sequences, the following oligonucleotideprobes are prepared.

P1: SEQ ID NO: 13

P2: SEQ ID NO: 14

P3: SEQ ID NO: 15

N1: SEQ ID NO: 16

N2: SEQ ID NO: 17

N3: SEQ ID NO: 18

N4: SEQ ID NO: 19

N5: SEQ ID NO: 20

Using the genomic DNA described previously as a template and aboveoligonucleotides as probes, hybridization is performed.

(3) Screening

The screening of a hexaprenyl diphosphate synthetase gene from thegenomic DNA of M. luteus B-P 26 may be performed by conventionalmethods, for example, Southern hybridization, colony hybridization, andthe like.

The present inventor has cloned an FPS gene of M. luteus B-P 26 by themethod described in Example 2 later. Accordingly, the location of theFPS gene can be easily identified by radio-labelling a DNA fragmentthereof designated B500 (SEQ ID NO: 28) and performing Southernhybridization. As seen in these results, the inventor has thought thatif there exist in the HexPS gene DNA sequences coding for the preservedamino acid sequences in prenyl diphosphate synthetases, those DNAfragments which are probe-positive and not derived from the FPS gene canbe selected.

Then, in the present invention, Southern hybridization of the genomicDNA of M. luteus B-P 26 was performed using the probes prepared asdescribed above.

Briefly, the genomic DNA of M. luteus B-P 26 is digested withappropriate restriction enzymes (EcoRI, HindIII, PstI) separately. Theresultant restriction fragments are electrophoresed on agarose gel.Then, the gel is treated with alkali to denature the DNA intosingle-stranded DNA, and is transferred to a nylon membrane. This nylonmembrane is UV-irradiated to fix the DNA on the membrane. Subsequently,hybridization is performed using a labeled probe or B500 as a probe.After washing, autoradiography is performed with a bio-image analyzer tothereby confirm the location of those DNA bands having homology to theprobe.

Probes (P1, P2, N3, N4 and N5) are end-labeled with ³² P byenzymatically transferring the phosphate group from [γ-³² P]ATP to the5' end. On the other hand, B500 is ³⁵ S-labeled with [α-³⁵ S]dCTP by therandom prime labeling method to obtain a probe.

(4) Cloning of DNA Fragments Weakly Hybridizing with Probe B500

The probe B500 is a DNA fragment containing a portion of the FPS gene ofM. luteus B-P 26, as described previously. Therefore, this probestrongly hybridizes with the FPS gene. Also, B500 contains DNA sequencescoding for the preserved amino acid sequences among prenyl diphosphatesynthetases. If the HexPS gene of interest has DNA sequences coding forthe preserved amino acid sequences among prenyl diphosphate synthetases,such DNA sequences also hybridize with B500.

Then, in order to screen a gene coding for the peptide of a prenyldiphosphate synthetase of the invention, the cloning of those DNAfragments weakly hybridizing with B500 other than those DNA fragmentsstrongly hybridizing with B500 because of the presence of the FPS geneis performed.

EcoRI-digested genomic DNA fragments 4-6 kbp in size are extracted fromagarose gel and inserted into pUC119. E. coli strain JM109 istransformed with the resultant plasmid to thereby prepare a DNA librarycovering this region. Then, colony hybridization is performed using eachprobe.

The resultant clones are cultured in an appropriate medium (e.g., LBliquid medium). Then, cells of each clone are harvested and disrupted bysonication, to thereby obtain a crude enzyme extract. HexPS activity isdetermined using this crude enzyme extract.

Thus, a clone containing the prenyl diphosphate synthetase gene isobtained, and this clone is used for the DNA sequencing.

(5) Determination of the DNA Sequence

The resultant clone is digested with an appropriate restriction enzymeand electrophoresed on agarose gel, to thereby prepare a restriction mapfrom the migration pattern and migration distance.

Based on this restriction map, the deletion of DNA fragment (i.e.,making into smaller fragments) is performed. Thus, the smallest cloneexhibiting activity is obtained, and the DNA sequence for the smallestclone exhibiting activity is analyzed.

The sequence may be determined using deletion clones which contain theinsert DNA having a deletion of about 200 bp at both ends in oppositedirections.

The screened clone is digested with an appropriate restriction enzyme(e.g., EcoRI, PstI) and cloned into a plasmid such as pUC119, pUC19 orthe like. Then, the DNA sequence of interest may be determined byconventional base sequence analysis methods such as the dideoxy methodby Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)). Thedetermination of the DNA sequence may be performed with an automaticbase sequence analyzer using such as T7 Sequencing Kit (Pharmacia).

Once the DNA sequence is thus determined, the DNA of interest can beobtained by hybridization using a DNA fragment obtained from such aschemical synthesis or PCR.

The DNA of the invention may be used as a gene to express a prenyldiphosphate synthetase.

As the thus determined DNA sequence for a DNA coding for the polypeptideof a subunit of the prenyl diphosphate synthetase, the base sequenceshown in SEQ ID NO: 21 may be given, for example. This base sequencecontains 3 open reading frames (ORFs) designated hex1, hex2 and hex3.These ORFs correspond to positions 216-644 (SEQ ID NO: 3), positions622-1359 (SEQ ID NO: 29) and positions 1368-2342 (SEQ ID NO: 4),respectively, in the DNA sequence shown in SEQ ID NO: 21.

The ORFs described above may be cloned as a whole or cloned individuallyby conventional recombinant DNA techniques. For example, the DNA codingfor the polypeptide of a subunit of the prenyl diphosphate synthetasemay be digested with an appropriate restriction enzyme to therebygenerate three DNA fragments each containing hex1, hex2 or hex3, whichare individually ligated to a plasmid vector digested with the samerestriction enzyme. Thus, individual ORFs may be cloned.

(6) Identification of Prenyl Diphosphate Synthetase Genes

In order to examine whether the three polypeptides obtained by theexpression of the three genes described above have activity or not,plasmids each containing one of the ORFs are prepared and a host cell istransformed with each of the plasmids. The resultant transformant iscultured to thereby prepare a crude enzyme extract. Then the enzymeactivity of prenyl diphosphate synthetase is examined using this crudeextract.

The transformation of a host cell with a recombinant vector may beperformed by, for example, adding a recombinant vector to competentcells prepared with CaCl₂, MgCl₂ or RbCl (when the host cell is E.coli).

In order to detect cells containing the gene of interest, the colony orplaque hybridization method (J. Sambrook, E. F. Fritsch and T. Maniatis,Molecular Cloning, Cold Spring Harbor Laboratory Press) usingoligonucleotide probes chemically synthesized based on the amino acidsequence for the protein, or the like may be used.

The fragments containing the thus cloned DNAs coding for thepolypeptides of the prenyl diphosphate synthetase may be individuallyre-incorporated into an appropriate vector (e.g., pMalc2, pTrc99A) tothereby express the genes highly in E. coli cells. Further, byre-incorporating the fragment into an appropriate vector, it is possibleto transform other procaryotic or eucaryotic host cell with the vector.Further, by introducing into such a vector an appropriate promoter andsequences involved in phenotypic expression, the gene can be expressedin each host cell.

As a host cell, mammal-derived cells such as COS cells, Chinese hamsterovary (CHO) cells, HELA cells (human cervical carcinoma cells), mouseSertoli's cells; insect-derived cells; and yeast-derived cells such asPichia pastolis and Saccharomyces cerevisiae may be enumerated. As avector to transform these cells, BacPAK6, pSVL, SV40 and the like may beenumerated. These vectors contain a replication origin, selectablemarker, promoter, polyadenylation signal and the like.

As a promoter for gene expression, a promoter from retrovirus, polyomavirus or the like may be used.

As a replication origin, one from SV40, polyoma virus, adenovirus, VSV,etc. may be used. As a selectable marker, a thymidine kinase gene, adihydrofolate reductase gene or the like may be used.

(7) Expression of Polypeptides

In order to accumulate the polypeptide of subunit (A) and/or thepolypeptide of subunit (B) of a prenyl diphosphate synthetase in aculture using the above-mentioned host-vector system and to collect thepolypeptide(s), the host cell is transformed with a recombinant DNAobtained by incorporating the gene of interest in an appropriate site inthe vector and then the resultant transformant is cultured. Further, inorder to separate and purify the peptide(s) from cells or culturesolution, known techniques may be used. For example, salting out, gelfiltration, ion exchange chromatography, affinity chromatography,reversed phase chromatography, and the like may be used for purificationindependently or in combination. Whether the purified polypeptide is thepolypeptide of interest or not may be confirmed by SDS polyacrylamidegel electrophoresis, Western blotting, or the like.

(8) Production of a Heterodimeric, Active Type Prenyl DiphosphateSynthetase

According to the present invention, an active type prenyl diphosphatesynthetase can be produced by preparing the peptides of the individualsubunits of a heterodimeric prenyl diphosphate synthetase by recombinantDNA techniques and mixing the resultant peptides of the individualsubunits.

For example, as subunit (A) of a prenyl diphosphate synthetase, theexpression product of hex1 may be given, and as subunit (B) theexpression product of hex3. By mixing the expression product of hex1 andthe expression product of hex3, an active type prenyl diphosphatesynthetase having high enzyme activity can be obtained.

It should be noted that a mixture of the expression product of hex1(subunit (A)) and the expression product of hex2 nor a mixture of theexpression product of hex3 (subunit (B)) and the expression product ofhex2 does not have the enzyme activity of prenyl diphosphate synthetase.

According to the present invention, an active type enzyme on which aspecific property has been conferred can be produced by preparingseparately the polypeptides of the two subunits of a heterodimericenzyme, the polypeptides being derived from different organisms and oneof the polypeptides having the specific property, and mixing theresultant polypeptides of these subunits.

The term "specific property" used herein means a specific property otherthan the enzymatic activity of the enzyme conferred on the enzymeadditionally. For example, thermal resistance, alkali resistance, acidresistance, long term storage stability, resistance to organic solvents,and the like may be enumerated.

In the present invention, increase in specific activity can also beachieved in addition to those specific properties.

As a specific example for such an enzyme, a heterodimeric, active typeprenyl diphosphate synthetase having a conferred thermal resistance andenhanced activity may be given which is obtained by mixing equal amountsof a polypeptide of a subunit of a prenyl diphosphate synthetase fromBacillus subtilis (ATCC 6633) represented substantially by the aminoacid sequence of SEQ ID NO: 5 and a polypeptide of a subunit of athermoresistant prenyl diphosphate synthetase from Bacillusstearothermophilus (ATCC 10149) represented substantially by the aminoacid sequence of SEQ ID NO: 6 (having thermal resistance).

PREFERRED EMBODIMENTS OF THE INVENTION

Hereinbelow, the present invention will be described in more detail withreference to the following Example, which should not be construed aslimiting the technical scope of the invention.

EXAMPLE 1 Preparation of Genomic DNA from Micrococcus luteus

M. luteus B-P 26 was cultured in 6 liters of L-broth (containing 10 g ofbacto tryptone, 5 g of bacto yeast extract, 5 g of NaCl and 1 g ofglucose per liter) at 30° C. for 24 hours. The culture solution wascentrifuged at 7,000 rpm at 4° C. for 15 minutes. The cells weresuspended and washed in saline-EDTA (0.15 M NaCl, 0.1 M EDTA, pH 8.0)and then re-centrifuged at 7,000 rpm at 4° C. for 10 minutes.

The cells thus obtained (about 17 g in wet weight) were suspended in 17ml of saline-EDTA. Then, 1 g of lysozyme was added to the cellsuspension and incubated at 37° C. for 30 minutes.

Subsequently, 36 ml of sterilized water, 36 ml of 1 M Tris-HCl buffer(pH 9.0), 12 ml of 5 M NaCl and 12 ml of 10% SDS were added to the cellsuspension and suspended well. Then, the resultant suspension was frozenusing liquid nitrogen. Thereafter, the frozen suspension was thawed at60° C. and 1.0 g of lysozyme was added thereto at room temperature.Then, the suspension was re-frozen at -70° C. and re-thawed at 60° C .This freezing-and-thawing was repeated again. The suspension wasincubated at 37° C. for 1 hour, and then 400 mg of lysozyme of 3.1 mg ofproteinase K were added thereto and incubated at 55° C. for 30 minutes.An equal volume (about 150 ml) of phenol was added thereto and agitatedslowly for 20 minutes while ice-cooling. The resultant suspension wascentrifuged at 3,000 rpm at 4° C. for 10 minutes and the supernatant wascollected. This supernatant was further centrifuged at 3,000 rpm at 4°C. for 30 minutes. The resultant supernatant was dispensed in 15 mlportions into 50 ml conical tubes (Falcon). Two volumes of ethanol wasadded to each tube gently and mixed gently. The white precipitate ofthread-like DNA was wound around a glass rod and dissolved in 36 ml ofdiluted saline-citrate (0.015 M NaCl, 0.0015 M trisodium citrate), towhich 4 ml of concentrated saline-citrate (1.5 M NaCl, 0.15 M trisodiumcitrate) was added. From the measurement of absorbance (A260), the crudeyield was found to be about 44 mg. In order to remove RNA, the followingoperations were performed. To the DNA solution, 40 ml of dilutedsaline-citrate was added to give a nucleic acid concentration of about0.5 mg/ml. Then, RNaseTI and RNaseA were added to give finalconcentrations of 3.6 μg/ml and 50 μg/ml, respectively, and incubated at37° C. for 30 minutes. An equal volume of phenol was added thereto andagitated slowly by turning the tube up and down for 10 minutes whileice-cooling the tube. The resultant phenol mixture was centrifuged at3,000 rpm at 4° C. for 10 minutes, and the aqueous layer was mixed witha mixed solution of phenol and chloroform (1:1).

The resultant solution was centrifuged at 3,000 rpm at 4° C. for 15minutes, and the aqueous layer was collected. Two volumes of ethanol wasadded to the aqueous layer to precipitate DNA. After centrifuged at15,000 rpm at 4° C. for 20 minutes, the precipitate was washed with 70%,80% and 90% ethanol in this order and finally suspended in 50 ml of TE(10 ml Tris-HCl, 1 mM EDTA, pH 7.4).

From the measurement of absorbance, the yield was found to be 4.95 mg.

EXAMPLE 2 Amplification of FPP Synthetase Gene Fragment (B500) by PCRand Cloning of FPP Synthetase Gene

The primers described below were synthesized based on the amino acidsequences of "Gly Gly Lys Arg Ile Arg Pro Leu" (SEQ ID NO: 7) inpreserved Region I, "Ser Leu Ile His Asp Asp" (SEQ ID NO: 8) and "AspLeu Arg Arg Gly Arg Pro" (SEQ ID NO: 9) in preserved Region II, "Leu AlaGly Asp Gly Leu Leu" (SEQ ID NO: 10) in preserved Region III, "Phe GlnIle Arg Asp Asp Ile Leu Asp" (SEQ ID NO: 11) and "Gly Lys Pro Val GlySer Asp" (SEQ ID NO: 12) in preserved Region VI found among prenyldiphosphate synthetases beyond difference in organism species.

Sense Primers:

P1: SEQ ID NO: 13

P2: SEQ ID NO: 14

P3: SEQ ID NO: 15

Antisense Primers:

N1: SEQ ID NO: 16

N2: SEQ ID NO: 17

N3: SEQ ID NO: 18

N4: SEQ ID NO: 19

N5: SEQ ID NO: 20

A PCR was performed using the genomic DNA described above as a templateand the above oligonucleotides as primers.

The PCR was performed in a PCR solution having the composition describedbelow 5 cycles, 1 cycle being at 97° C. for 90 seconds, at 40° C. for 90seconds and at 72° C. for 120 seconds, followed by 20 cycles, 1 cyclebeing at 96° C. for 90 seconds, at 55° C. for 90 seconds and at 72° C.for 120 seconds.

    ______________________________________                                        Composition of the PCR Solution:                                              ______________________________________                                        Genomic DNA       1          μg                                            Tris-HCl (pH 8.3) 10         mM                                               KCl               50         mM                                               MgCl.sub.2        1.5        mM                                               Gelatin           0.001%     (w/v)                                            dNTP mixture      200        μM each                                       Primers           0.1        nmol each                                        Ampli Taq DNA Polymerase                                                                        2.5        u                                                (Total volume     100        μl)                                           ______________________________________                                    

An approx. 500 bp band (B500) which is specifically amplified with acombination of P1 and N3 was obtained. This DNA fragment was ligated topT7Blue T-vector (Novagen) to thereby obtain pB500. As a result of thedetermination of its DNA sequence by the dideoxy method, it was foundthat the amino acid sequence encoded by B500 has 60.7% homology to theamino acid sequence of FPS from Bacillus stearothermophilus in 145 aminoacids. The genomic DNA from M. luteus B-P 26 was partially digested withSau3AI. The resultant DNA fragments of 4-8 kbp were inserted intopUC119/BamHI, and E. coli strain JM109 was transformed with theseplasmids to thereby prepare a genomic library. pB500 was digested withPstI and BamHI and then electrophoresed on agarose gel to thereby cutoff and recover DNA fragments. The B500 fragments obtained were labeledusing Random Primer Labeling Kit (Takara Shuzo) according the protocolattached thereto. A library of about 6000 colonies was screened bycolony hybridization with labeled B500 fragments as probes to therebyobtain clones hybridizing with B500. The prenyl diphosphate synthetaseactivity of these clones was measured and their products were analyzed.As a result, one product was confirmed to be an FPP synthetase.

EXAMPLE 3 Southern Hybridization of the Genomic DNA from M. luteus B-P26

(1) Blotting of the Genomic DNA to a Nylon Membrane

The genomic DNA from M. luteus B-P 26 prepared in Example 1 wassubjected to Southern blotting.

    ______________________________________                                        Genomic DNA   100          μl (10 μg)                                   10x Buffer    30           μl                                              Sterilized water                                                                            162          μl                                              Enzyme        8            μl                                              ______________________________________                                    

In the above composition, 10 μg of the genomic DNA from M. luteus B-P 26was reacted with one of the restriction enzymes EcoRI(10 U/μl), PstI(100 U/μl) and HindIII (12 U/μl) at 37° C. for 40 hours to allowcomplete digestion. The reaction solution was treated withphenol-chloroform and then with chloroform. Thereafter, the solution wasethanol-precipitated. The resultant DNA was dried under reducedpressured and then dissolved in 100 μl of TE (10 mM Tris-HCl, 1 mM EDTA,pH 8.0) at a concentration of 350 ng/μl. Three DNA solutions (10 μleach) were electrophoresed on 0.8% agarose gel. For Southern blotting, aplate transfer device, NA-1512 (Nippon Eido K.K.), was used. Therestriction enzymes used were commercial enzymes (available from TakaraShuzo, NEB or Boehringer Mannheim).

The agarose gel after the electrophoresis of DNA was soaked in asolution containing 1.5 M NaCl and 0.5 M NaOH and shaken slowly for 30minutes, to thereby denature the DNA. A nylon membrane and a filterpaper were cut into the same size as that of the gel and soaked in asolution containing 0.25 M NaOH and 1.5 M NaCl. The agarose gel and thenylon membrane were layered in the plate transfer device as shown inFIG. 3. Then, an electric current (constant) of 150 mA was applied tothe device for 60 minutes to thereby transfer the DNA onto the nylonmembrane. This membrane was washed with 5×SSC (0.5 M NaCl, 0.075 Msodium citrate), air-dried on a filter paper and then irradiated with UVrays (120 mJ/cm²), to thereby fix the DNA on the membrane.

The thus obtained nylon membrane was used in Southern hybridization.

(2) Preparation of Probes for Southern Hybridization

B500, which is a DNA fragment from the FPS gene, was labeled usingrandom primers (9 mers) and [α-³⁵ S]dCTP (Amersham). The syntheticoligonucleotides used as probes were labeled by enzymaticallytransferring to its 5' end the phosphate group at γ position of [γ-³²P]ATP (Amersham) by means of T4 polynucleotide kinase contained in thefollowing composition.

    ______________________________________                                        5'-OH oligonucleotides (P1, P2, N3, N4, N5)                                                          10       pmol                                          10x Kinase buffer      1        μl                                         [γ-.sup.32 P]ATP (3000 Ci/mmol, 10 μCi/μl)                                               3        μl                                         T4 polynucleotide kinase (10 U/μl)                                                                1        μl                                         Sterilized water to make                                                                             10       μl                                         10x Kinase buffer: 0.5M Tris-HCl (pH 8.0)                                     0.1M MgCl.sub.2                                                               50 mM DTT solution                                                            ______________________________________                                    

The above composition was reacted at 37° C. for 30 hours, and thenheat-treated at 95° C. for 3 hours to inactivate T4 polynucleotidekinase. The thus obtained end-labeled oligonucleotides were used asprobes for Southern hybridization.

Subsequently, hybridization with the labeled probes and washing wereperformed. Then, autoradiography was performed using a bio-imageanalyzer manufactured by Fiji Film.

Three identical nylon membrane were prepared and designated No. 1, No. 2and No. 3. First, No. 1 was hybridized with probe B500, No. 2 with probeN4, and No. 3 with probe N5. Then, autoradiography was performed.Thereafter, the filters were washed to remove the hybridizing probescompletely. Thereafter, they were re-hybridized with probes P1, P2 andN3, respectively, and then autoradiographed.

The results are shown in FIG. 4. From these autoradiograms, those bandsindicated with mark "▴" appear to be fragments from the FPS gene in viewof the strong binding to probe B500. Further, with respect to thegenomic DNA digested with EcoRI, a weak band (indicated with mark "Δ";approx. 4.2 kbp) was confirmed below the FPS band. It is seen that thisband of approx. 4.2 kbp is also bound to probe N4 relatively strongly.

Since the FPS gene does not have an EcoRI restriction site in itsinside, the FPS gene is never digested with EcoRI to exhibit two bands.Accordingly, there is a possibility that this weakly binding, approx.4.2 kbp band contains a gene of other prenyl diphosphate synthetase.

Therefore, this DNA fragment of approx. 4.2 kbp was cloned.

(3) Cloning of the DNA Fragment Weakly Hybridizing with Probe B500

4-6 kbp fractions of the EcoRI-digested genomic DNA were cut off fromagarose gel and DNA fragments were recovered using The GENECLEAN II Kit(BIO 101).

These DNA fragments were inserted into pUC119 and then E. coli strainJM109 was transformed with the plasmid. Subsequently, colonyhybridization was performed.

As a result, three clones hybridizing with every probe were obtainedfrom about 1,200 colonies. These clones were cultured in LB medium. Thecells were harvested and disrupted by sonication, to thereby obtain acrude enzyme extract. The measurement of prenyl diphosphate synthetaseactivity was performed on this crude enzyme solution.

The measurement of activity was performed as follows.

Briefly, positive colonies were cultured in 50 ml of L medium overnight.Then, cells were harvested by centrifugation at 3,000 rpm at 4° C. for20 minutes and suspended in 3 ml of TE. This suspension was sonicated todisrupt cells. Then, the suspension was centrifuged at 3,000 rpm at 4°C. for 20 minutes to obtain a supernatant. The supernatant was furthercentrifuged at 15,000 rpm for 5 minutes to thereby obtain a supernatantas a crude enzyme extract. Sonication was performed at an output of 40Wwith 30% pulse for 5 minutes.

    ______________________________________                                        Crude homogenate 20          μg                                            Tris-HCl (pH 7.5)                                                                              100         mM                                               MgCl.sub.2       5           mM                                               FPP              5           μM                                            [1-.sup.14 C]IPP (54 Ci/mol)                                                                   0.46        μM                                            H.sub.2 O to make                                                                              1           ml                                               ______________________________________                                    

The above composition was reacted at 37° C. for 3 hours. To the reactionsolution, 3 ml of butanol was added and agitated. The mixture was leftstationary or centrifuged to thereby separate into two layers. Thebutanol layer was recovered. Using 500 μl of this layer, the level ofradioactivity in the product was measured with a liquid scintillationcounter. Also, 2 ml of the recovered butanol layer was treated with acidphosphatase and subjected to reversed phase thin layer chromatography(TLC), to thereby analyze the product.

    ______________________________________                                        BuOH extract       2           ml                                             Acetate buffer (pH 5.6)                                                                          1           ml                                             5% Triton X-100    100         μl                                          MeOH               1           ml                                             Acid phosphatase (1 g/28 ml)                                                                     500         μl                                          ______________________________________                                    

The above composition was reacted at 37° C. for 14 hours. To thereaction solution, 4 ml of pentane was added and agitated. The mixturewas left stationary or centrifuged to thereby separate into two layers.Then, the pentane layer was recovered. The solvent was removed withnitrogen gas, and the remaining material was dissolved in 100 μl ofpentane and applied to LKC18 reversed phase thin layer chromatography(Whatman; developing solvent: acetone/water=19/1).

The results are shown in FIG. 5.

From these results, the product was found to be a HexPP.

Thus, a clone containing a HexPP gene was obtained and designated pHX00.Hereinafter, this clone was used in the analysis of DNA sequences.

EXAMPLE 4 Analysis of a Restriction Map and DNA Sequences

(1) Preparation of Deletion Clones from pHX00

A restriction map of pHX00 was created and deletion clones were preparedbased on the map. Thus, DNA fragments of different length were obtained(FIG. 6). As shown in FIG. 6, fragments pHX01-pHX05 and pEcoRV-L wereprepared as follows. First, pHX00 was digested with XbaI, and the digestwas cut at the site indicated for each fragment (e.g., MluI site forpHX02 and NruI site for pHX05). The resultant digest was electrophoresedon agarose gel and a DNA fragment having the predicted length wasrecovered using The GENECLEAN II Kit (BIO 101). Subsequently, the endsof the DNA fragment were blunted with T4 DNA polymerase. Then, adeletion clone of interest was prepared by self ligation.

Fragments pSacI-L and pSacI-s in FIG. 6 represent the two fragmentsgenerated by cutting pHX00 with SacI. The longer fragment (containingpUC119) was self-ligated to obtain pSacI-s, and the shorter fragment wasligated to another pUC119 digested with SacI to obtain pSacI-L.

Fragments pPstI-L and pPstI-s in FIG. 6 were obtained in a similarmanner from pHX01. In other words, pHX01 was digested with PstI. Thelonger one of the resultant two fragments was self-ligated to obtainpPstI-s and the shorter one was inserted into the PstI site of anotherpUC119 to obtain pPstI-L (FIG. 6).

With pSacI-L and pPstI-L, two plasmids of opposite directions ofinsertion are obtained for each of them. These plasmids of oppositedirections were designated pSacI-L-1 and -2 and pPstI-L-1 and -2,respectively.

For each of the deletion clones described above, prenyl diphosphatesynthetase activity was measured. As a result, a clone of the minimumlength exhibiting activity, pHX05, was obtained (FIG. 6).

Further, deletion clones having serial deletions in the right and theopposite direction of the gene were prepared from pPstI-L-1 andpPstI-L-2 using Deletion Kit for Kilo-sequence (Takara Shuzo) accordingto the protocol attached to the Kit. Using the thus prepared deletionclones, base sequences were analyzed.

(2) Determination of the DNA Sequence

The deletion clones prepared by the alkali-SDS method wasalkali-denatured as described below to prepare a template DNA.

    ______________________________________                                        Template     32        μl (1.5-2 μg DNA)                                2M NaOH      8         μl                                                  Total volume 40        μl                                                  ______________________________________                                    

The above solution was prepared, agitated gently, and incubated at roomtemperature for 10 minutes. To the resultant solution, 7 μl of 3 Msodium acetate (pH 4.8) and 4 μl of distilled water were added.

Then, 120 μl of ethanol was added further and mixed. Thereafter, thesolution was placed in dry ice for 15 minutes. The solution wascentrifuged at 15,000 rpm at 4° C. for 15 minutes to precipitate theDNA, which was washed with 70% ethanol and centrifuged at 15,000 rpm at4° C. for 10 minutes. The supernatant was discarded. Then, the DNA wasdried under reduced pressure and dissolved in 10 μl of distilled water.

Using the thus prepared DNA as a template DNA, analysis of the basesequence was performed by the dideoxy method with T7 Sequencing Kit(Pharmacia) and [α-³⁵ S]dCTP (Amersham).

The DNA sequence analyzed is shown in SEQ ID NO: 21.

As a result, it has become clear that pHX05 contains 3 open readingframes coding for proteins. They were designated hex1, hex2 and hex3from the upstream (FIG. 7).

hex1 codes for a protein composed of 143 amino acids shown in SEQ IDNO: 1. The presumed molecular weight of this protein was 17 kDa. hex2codes for a protein composed of 246 amino acids shown in SEQ ID NO: 29.The presumed molecular weight of this protein was 28 kDa. The first 23bases including the initiation codon overlapped with a downstreamportion of hex1. hex3 codes for a protein composed of 325 amino acidsshown in SEQ ID NO: 2. The presumed molecular weight of this protein was37 kDa.

The amino acid sequences (Hex1, Hex2 and Hex3) of the proteins encodedby these 3 open reading frames (ORFs) were compared with the amino acidsequences (Hep1 and Hep2) of the proteins encoded by HepPS genes (heP1,hep2) of Bacillus stearothermophilus. As a result, Hex3 exhibits 36.4%homology to Hep2 and retains the 7 preserved regions describedpreviously common in prenyl diphosphate synthetases.

On the other hand, Hex1 exhibits only 8.4% homology to Hep1 and iscomposed of a very small number (i.e., 143) of amino acids whereas Hep1is composed of 220 amino acids.

The structural genes of the Bacillus stearothermophilus HepPS are hep1and hep2. From the homology to these genes, hex3 was expected to be thestructural gene of the HexPS of M. luteus B-P 26. However, hex1 and hex2could not be judged so because they do not exhibit high homology tohep1. Then, the inventor decided to confirm which ORF is the structuralgene of the HexPS.

EXAMPLE 5 Measurement of the Activity of Heterodimeric PrenylDiphosphate Synthetases (1)

(i) Identification of the HexPP Synthetase Gene

In order to prepare a plasmid containing hex1 alone, pde1 2-5 wasdigested with HincII and NruI and the resultant fragments wereself-ligated (FIGS. 8 and 9). Thus, pREG1 was obtained. Similarly, inorder to prepare a plasmid containing hex2 alone, SacI-SacI fragmentfrom pde1 1-1 was cut off and inserted into the SacI site of pUC119, tothereby obtain pREG2 (FIGS. 8 and 9).

A plasmid containing hex3 alone was prepared as follows. pde11-13 wasdigested with EcoRI. After the fragments were blunt-ended, they werefurther digested with PstI. Further, pPstI-S was digested with HindIII.After the fragments were blunt-ended, they were digested with PstI.These fragments were ligated to obtain pREG3. Since pREG3 contains about400 bp of hep3 located in its downstream, pREG3 was digested with EcoRIand EcoT14I, and after the ends of the fragments were blunted, thefragments were ligated to obtain pREG3S (FIGS. 8 and 9). "pde1 2-5","pde1 1-1" and "pde1 1-13" used herein are the deletion clones ofpPstI-L described previously which were prepared with Deletion Kit forKilo-Sequence.

Thus, plasmids each containing one of the three regions (hex1, hex2 andhex3) found in the insert DNA of pHX05 were prepared (FIG. 9).

Since pHX05 containing hex1, hex2 and hex3 includes about 400 bp of hex3located in its downstream, pHX00 was digested with NruI and EcoT14I andthen ligated to HincII-digested pUC119. Of the resultant two clones, onehaving the same direction of gene as that of pHX05 was selected anddesignated pHX06 (FIG. 9).

E. coli strain JM109 was transformed with above plasmids and enzymeactivity was measured as described previously.

Transformants carrying plasmids pREG1, pREG2 and pREG3 containing thehex1, hex2 and hex3 genes, respectively, have been deposited with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology as follows:

    ______________________________________                                                     Accession Number                                                 ______________________________________                                        pREG1/JM109    FERM BP-5910                                                   pREG2/JM109    FERM BP-5911                                                   pREG3S/JM109   FERM BP-5912                                                   ______________________________________                                    

(ii) Measurement of Enzyme Activity

A crude enzyme extract was prepared from each of the clones obtained.The enzyme activity of the combinations shown in Table 1 was examined.

The results are shown in table 1 and FIG. 10.

                  TABLE 1                                                         ______________________________________                                        M. luteus                                                                     Lane Homogenate      Activity (dpm)                                           ______________________________________                                        1. JM109/pHX05*      2693                                                     2. JM109/pHX06*      2533                                                     3. JM109/vector*       0                                                      4. JM109/pREG1 + JM109/vector                                                                       53                                                      5. JM109/pREG2 + JM109/vector                                                                        0                                                      6. JM109/PREG3S + JM109/vector                                                                       0                                                      7. JM109/pREG1 + JM109/pREG2                                                                         0                                                      8. JM109/PREG2 + JM109/PREG3S                                                                        0                                                      9. JM109/pREG1 + JM109/PREG3S                                                                      2924                                                     ______________________________________                                         1 μg each, *: 2 μg                                                 

Lanes 1-9 in FIG. 10 correspond to lanes 1-9 in Table 1.

From Table 1 and FIG. 10, it has been found that HexPS activity isexhibited when the polypeptides encoded by hex1 and hex2 are present atthe same time (Table 1, lanes 1, 2 and 9: FIG. 10, lanes 1 and 9). Inother words, the enzyme activity is manifested by mixing polypeptideHex1 [i.e., polypeptide of subunit (A)] with polypeptide Hex3 [i.e.,polypeptide of subunit (B)]. From the above results, it is also shownthat hex1 and hex3 are the structural genes of the HexPS.

EXAMPLE 6 Measurement of the Activity of Heterodimeric PrenylDiphosphate Synthetases (2)

(i) Construction of Plasmids Containing a HepPS Gene from Bacillussubtilis

From data base search, it has been found that genes exhibiting a highhomology to hep1 and hep2 (which are HepPS genes of Bacillusstearothermophilus) exist in Bacillus subtilis. One corresponding tohep1 is gerC1 and the other corresponding to hep2 is gerC3. Based on theDNA sequences registered in GenBank M80245, the followingoligonucleotides were synthesized and used as PCR primers.

    ______________________________________                                                   Sense primers                                                                 P6': SEQ ID NO: 22                                                            P5': SEQ ID NO: 23                                                            Antisense primers                                                             P2': SEQ ID NO: 24                                                            P4': SEQ ID NO: 25                                                 ______________________________________                                    

Bacillus subtilis (ATCC 6633) was cultured in 1 liter of LB medium(containing 10 g of bacto tryptone, 5 g of bacto yeast extract and 10 gof NaCl per liter) at 37° C. until OD600 reached 1. The culture solutionwas centrifuged at 7,000 rpm at 4° C. for 15 minutes and the cells wereharvested.

Genomic DNA from Bacillus subtilis was prepared according to the methodemployed in preparing genomic DNA from Bacillus stearothermophilus(Koike-Takeshita, A. et al. (1995) J. Biol. Chem., 270, 18396) and usedas a template for PCR.

A PCR was performed using the above genomic DNA as a template and usingthe oligonucleotides described above as primers.

The PCR was performed 24 cycles in a PCR solution having the compositiondescribed below, 1 cycle being at 74° C. for 30 seconds, at 55° C. for60 seconds and at 72° C. for 60 minutes. Then, the solution was heatedto 72° C. and reacted for 7 minutes.

    ______________________________________                                        Composition of the PCR Solution:                                              ______________________________________                                        Genomic DNA          1        μg                                           10x Ampli Taq DNA Polymerase buffer                                                                10       μl                                           dNTP mixture         200      μM each                                      Primers              0.2      μM each                                      Ampli Taq DNA Polymerase                                                                           2.5      U                                               (Total volume        100      μl)                                          ______________________________________                                    

For a clone having gerC1, PCR was performed using a combination of P6'and P4' primers. The amplified DNA fragments were digested with NcoI andHindIII and ligated to pTrc99A digested with NcoI and HindIII to therebyprepare pEHA1.

For a clone having gerC3, PCR was performed using a combination of P5'and P2' primers. The amplified DNA fragments were digested with NcoI andBglII and ligated to pTrc99A digested with NcoI and BamHI to therebyprepare pEHA3.

(ii) Construction of Plasmids having hepPS Gene from a Bacillusstearothermo-philus (ATCC 10149)

As a clone having hep1, pTLD7 disclosed in Koike-Takeshita, A. et al.(1995) J. Biol. Chem., 270, 18396 was used.

A clone having hep2 was prepared as follows. PCR was performed usingpTL6 disclosed in Koike-Takeshita, A. et al. supra as a template and theoligonucleotide described below as primers. The amplified DNA fragmentswere digested with BspHI and HindIII and ligated to pTrc99A digestedwith NcoI and HindIII to thereby obtain pHE5.

    ______________________________________                                                  Sense primer                                                                  HPP10: SEQ ID NO: 26                                                          Antisense primer                                                              HPP12: SEQ ID NO: 27                                                ______________________________________                                    

(iii) Expression of Proteins

E. coli strain JM109 was transformed with the four plasmids pEHA1,pEHA3, pTLD7 and pHE5 described above. Transformants obtained with pEHA1and pEHA3 were cultured in M9YG medium (1×M9 salt, 0.2% glycerol, 0.2%yeast extract), and transformants obtained with pTLD7 and pHE5 in LBmedium. In the late logarithmic growth phase, 1 mM IPTG was added toeach medium and cells were cultured for another 3 hours. Thereafter, theculture solution was centrifuged at 5,000 rpm for 20 minutes to therebyharvest cells. Then, about 0.2 g of cells from each transformant weresuspended in 1 ml of lysis buffer (25 mM Tris-HCl, 1 mM EDTA, 10 mM2-mercaptoethanol) and sonicated to disrupt cells. Then, the cellsuspension was centrifuged at 15,000 rpm for 5 minutes to obtain thesupernatant as a crude enzyme solution. Only those supernatants fromtransformants obtained with pTLD7 and pHE5 were heat-treated at 55° C.for 15 minutes.

(iv) Measurement of Enzyme Activity

The proteins expressed were mixed in equal amounts in the combinationsshown in Table 2 below. Then, HepPS activity of each combination wasexamined.

The following composition was reacted at 37° C. for 1 hour. The reactionproduct was extracted with butanol as described previously and used forthe measurement of radioactivity and analysis by reversed phase TLC.

    ______________________________________                                        Crude homogenates                                                                              1          μg each                                        Tris-HCl (pH 8.0)                                                                              50         mM                                                MgCl.sub.2       1          mM                                                NH.sub.4 Cl      50         mM                                                2-Mercaptoethanol                                                                              50         mM                                                FPP              25         μM                                             [1-.sup.14 C]IPP (54 Ci/mol)                                                                   0.46       μM                                             ______________________________________                                    

A combination of polypeptides GERC1+GERC3 and another combination ofpolypeptides GERC1+Hep2 exhibit prenyl diphosphate synthetase activity(Table 2). It was confirmed from the analysis of the reaction productsthat both combinations synthesize HepPP (FIG. 11) .

                  TABLE 2                                                         ______________________________________                                        Activity of Hybrid Enzymes from B. subtilis                                   and B. stearothermophilus                                                     Homogenate      Activity (dpm)                                                ______________________________________                                        GERC1 + GERC3   136                                                           GERC1 + Hep2    7919                                                          GER1 + GERC3     0                                                            Hep1 + Hep2      0                                                            ______________________________________                                         Protein Amount of homogenate: 1 μg each                               

(v) Comparison of Thermal Stability

Polypeptides GERC1 and GERC3 are derived from Bacillus subtilis which isa mesophile. Polypeptide Hep2 is derived from Bacillusstearothermophilus which is a moderate thermophilic bacterium. Then, theoptimum reaction temperature and the thermal stability of HepPSsobtained from the combinations of GERC1+GERC3 and GERC1+Hep2,respectively, were compared.

The combination of GERC1+GERC3 and the combination of GERC1+Hep2 werereacted separately at 10, 15, 20, 25, 30, 37, 40, 45, 50, 55 and 60° C.and the radioactivities of the butanol extracts were compared (FIG. 12).While the optimum reaction temperature of the HepPS resulted from thecombination of GERC1+GERC3 is 25° C., that of the HepPS resulted fromthe combination of GERC1+Hep2 has been raised to 40° C., showing adifference of 15° C.

Further, the residual activity of both combinations after heat treatmentwas compared. Crude enzyme solutions were mixed for each combination andheat-treated at 37, 45, 50 and 55° C. for 20 minutes. Thereafter, thereaction solution was reacted at 30° C. for 1 hour and extracted withbutanol. The radioactivities of the butanol extracts were compared (FIG.13).

While the residual activity of the HepPS from GERC1+GERC3 is 72% after37° C. treatment, 20% after 45° C. treatment and 7% after 50° C.treatment, the residual activity of the HepPS from GERC1+Hep2 is 102%after 45° C. treatment and 83% after 50° C. treatment.

Although it is known that component II (which appears to correspond toGERC3 of the HepPS from Bacillus subtilis) is less stable against heatthan component I (which appears to correspond to gerC1) (Fujii, H. etal., (1983) FEBS Lett., 161, 257), the construction of a hybrid enzymebetween GERC1 from Bacillus subtilis and Hep2 (corresponding tocomponent II) from the moderate thermophilic bacterium Bacillusstearothermophilus has rendered thermal stability on this enzyme.

EFFECT OF THE INVENTION

According to the present invention, there are provided a method forproducing peptides of prenyl diphosphate synthetases, a method forproducing an active type prenyl diphosphate synthetase, a DNA coding forthe synthetase, a recombinant vector comprising the DNA and atransformant comprising the vector.

Substances synthesized by the prenyl diphosphate synthetase of theinvention are precursors of those substances such as vitamin K andubiquinones which exist universally in organisms and, thus, they areimportant physiologically active substances. Therefore, they are of highutility value. Furthermore, the prenyl diphosphate produced by aheterodimeric prenyl diphosphate synthetase is extremely useful sincethe chain length and structural isomers thereof can be strictlycontrolled.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 29                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 143 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Arg Tyr Leu His Lys Ile Glu Leu Glu Le - #u Asn Arg Leu Thr Ser         #15                                                                           - Arg Tyr Pro Phe Phe Lys Lys Ile Ala Phe As - #p Ala Glu Ile Ile Lys         #             30                                                              - Leu Val Asp Asp Leu Asn Val Asp Glu Asn Va - #l Lys Cys Ala Ile Val         #         45                                                                  - Ala Ile Asp Thr Ser Met Arg Met Gln Asp Ph - #e Ile Asn Glu Asp Asn         #     60                                                                      - Lys Asp Ser Phe Val Leu Ser Thr Asp Val Le - #u Ser Ala Leu Phe Tyr         #805                                                                          - Lys Tyr Leu Ser Gln Pro Phe Tyr Gln His As - #p Phe Leu Val Leu Thr         #                 95                                                          - Asp Cys Val Ser Arg Ile Asn Glu Leu Lys Se - #r Ile Arg Ala Thr Ile         #           110                                                               - Thr Asp Glu Ile Ala Leu His Asn Ile Asn Ly - #s Gln Ile His Tyr Met         #       125                                                                   - Phe Ile Gln Pro Tyr Met Asn Asn Glu Lys Va - #l Val Ser Tyr Glu             #   140                                                                       - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 325 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Ile Ala Leu Ser Tyr Lys Ala Phe Leu As - #n Pro Tyr Ile Ile Glu         #15                                                                           - Val Glu Lys Arg Leu Tyr Glu Cys Ile Gln Se - #r Asp Ser Glu Thr Ile         #             30                                                              - Asn Lys Ala Ala His His Ile Leu Ser Ser Gl - #y Gly Lys Arg Val Arg         #         45                                                                  - Pro Met Phe Val Leu Leu Ser Gly Phe Leu As - #n Asp Thr Gln Lys Asp         #     60                                                                      - Asp Leu Ile Arg Thr Ala Val Ser Leu Glu Le - #u Val His Met Ala Ser         # 80                                                                          - Leu Val His Asp Asp Tyr Ile Asp Asn Ser As - #p Met Arg Arg Gly Asn         #95                0                                                          - Thr Ser Val His Ile Ala Phe Asp Lys Asp Th - #r Ala Ile Arg Thr Gly         #           110                                                               - His Phe Leu Leu Ala Arg Ala Leu Gln Asn Il - #e Ala Thr Ile Asn Asn         #       125                                                                   - Ser Lys Phe His Gln Ile Phe Ser Lys Thr Il - #e Leu Glu Val Cys Phe         #   140                                                                       - Gly Glu Phe Asp Gln Met Ala Asp Arg Phe As - #n Tyr Pro Val Ser Phe         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Ala Tyr Leu Arg Arg Ile Asn Arg Lys Th - #r Ala Ile Leu Ile Glu         #               175                                                           - Ala Ser Cys His Leu Gly Ala Leu Ser Ser Gl - #n Leu Asp Glu Gln Ser         #           190                                                               - Thr Tyr His Ile Lys Gln Phe Gly His Cys Il - #e Gly Met Ser Tyr Gln         #       205                                                                   - Ile Ile Asp Asp Ile Leu Asp Tyr Thr Ser As - #p Glu Ala Thr Leu Gly         #   220                                                                       - Lys Pro Val Gly Ser Asp Ile Arg Asn Gly Hi - #s Ile Thr Tyr Pro Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Met Ala Ala Ile Ala Asn Leu Lys Glu Gln As - #p Asp Asp Lys Leu Glu         #               255                                                           - Ala Val Val Lys His Leu Thr Ser Thr Ser As - #p Asp Glu Val Tyr Gln         #           270                                                               - Tyr Ile Val Ser Gln Val Lys Gln Tyr Gly Il - #e Glu Pro Ala Glu Leu         #       285                                                                   - Leu Ser Arg Lys Tyr Gly Asp Lys Ala Lys Ty - #r His Leu Ser Gln Leu         #   300                                                                       - Gln Asp Ser Asn Ile Lys Asp Tyr Leu Glu Gl - #u Ile His Glu Lys Met         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Lys Arg Val Tyr                                                                         325                                                           - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 432 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION:1..429                                                 #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG CGT TAT TTA CAT AAA ATT GAA CTA GAA TT - #A AAC CGA CTT ACA AGT           48                                                                          Met Arg Tyr Leu His Lys Ile Glu Leu Glu Le - #u Asn Arg Leu Thr Ser           #15                                                                           - CGA TAT CCA TTT TTC AAA AAA ATT GCA TTT GA - #T GCT GAA ATC ATA AAG           96                                                                          Arg Tyr Pro Phe Phe Lys Lys Ile Ala Phe As - #p Ala Glu Ile Ile Lys           #             30                                                              - CTC GTT GAT GAC CTA AAT GTC GAT GAA AAT GT - #A AAA TGT GCG ATT GTT          144                                                                          Leu Val Asp Asp Leu Asn Val Asp Glu Asn Va - #l Lys Cys Ala Ile Val           #         45                                                                  - GCC ATT GAC ACG AGT ATG CGT ATG CAG GAT TT - #T ATC AAT GAA GAT AAT          192                                                                          Ala Ile Asp Thr Ser Met Arg Met Gln Asp Ph - #e Ile Asn Glu Asp Asn           #     60                                                                      - AAA GAC AGT TTT GTA CTA TCA ACG GAT GTT TT - #G AGT GCT TTA TTT TAT          240                                                                          Lys Asp Ser Phe Val Leu Ser Thr Asp Val Le - #u Ser Ala Leu Phe Tyr           # 80                                                                          - AAG TAT TTA TCA CAG CCA TTT TAT CAG CAT GA - #T TTT TTA GTA CTG ACG          288                                                                          Lys Tyr Leu Ser Gln Pro Phe Tyr Gln His As - #p Phe Leu Val Leu Thr           #                 95                                                          - GAT TGT GTA AGT CGT ATC AAT GAA TTA AAA TC - #A ATA AGA GCA ACG ATT          336                                                                          Asp Cys Val Ser Arg Ile Asn Glu Leu Lys Se - #r Ile Arg Ala Thr Ile           #           110                                                               - ACA GAC GAA ATT GCT TTG CAT AAT ATT AAT AA - #A CAA ATT CAT TAT ATG          384                                                                          Thr Asp Glu Ile Ala Leu His Asn Ile Asn Ly - #s Gln Ile His Tyr Met           #       125                                                                   - TTC ATA CAA CCT TAT ATG AAC AAT GAG AAA GT - #G GTG TCT TAT GAG TAA          432                                                                          Phe Ile Gln Pro Tyr Met Asn Asn Glu Lys Va - #l Val Ser Tyr Glu               #   140                                                                       - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 978 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION:1..975                                                 #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG ATT GCT TTG AGT TAT AAA GCG TTT TTA AA - #C CCA TAT ATC ATT GAA           48                                                                          Met Ile Ala Leu Ser Tyr Lys Ala Phe Leu As - #n Pro Tyr Ile Ile Glu           #15                                                                           - GTT GAA AAA AGG TTA TAT GAG TGT ATT CAG AG - #T GAT TCT GAA ACG ATA           96                                                                          Val Glu Lys Arg Leu Tyr Glu Cys Ile Gln Se - #r Asp Ser Glu Thr Ile           #             30                                                              - AAC AAG GCG GCA CAC CAT ATT TTA AGT TCA GG - #A GGA AAG CGC GTA CGT          144                                                                          Asn Lys Ala Ala His His Ile Leu Ser Ser Gl - #y Gly Lys Arg Val Arg           #         45                                                                  - CCG ATG TTT GTA TTA TTA AGT GGT TTT CTG AA - #T GAT ACA CAA AAG GAT          192                                                                          Pro Met Phe Val Leu Leu Ser Gly Phe Leu As - #n Asp Thr Gln Lys Asp           #     60                                                                      - GAC TTG ATT CGT ACA GCA GTA TCT CTG GAG CT - #C GTT CAT ATG GCA AGT          240                                                                          Asp Leu Ile Arg Thr Ala Val Ser Leu Glu Le - #u Val His Met Ala Ser           # 80                                                                          - CTC GTT CAT GAT GAT TAC ATC GAT AAT AGT GA - #T ATG CGT CGT GGT AAT          288                                                                          Leu Val His Asp Asp Tyr Ile Asp Asn Ser As - #p Met Arg Arg Gly Asn           #                 95                                                          - ACT TCG GTT CAT ATA GCT TTT GAT AAA GAC AC - #A GCA ATT CGC ACA GGA          336                                                                          Thr Ser Val His Ile Ala Phe Asp Lys Asp Th - #r Ala Ile Arg Thr Gly           #           110                                                               - CAT TTT TTA TTA GCA CGT GCG TTA CAA AAT AT - #T GCA ACT ATC AAT AAT          384                                                                          His Phe Leu Leu Ala Arg Ala Leu Gln Asn Il - #e Ala Thr Ile Asn Asn           #       125                                                                   - TCG AAA TTC CAT CAA ATT TTT AGT AAA ACG AT - #A CTT GAA GTT TGT TTT          432                                                                          Ser Lys Phe His Gln Ile Phe Ser Lys Thr Il - #e Leu Glu Val Cys Phe           #   140                                                                       - GGT GAA TTT GAC CAG ATG GCA GAT CGA TTT AA - #T TAT CCT GTA TCC TTT          480                                                                          Gly Glu Phe Asp Gln Met Ala Asp Arg Phe As - #n Tyr Pro Val Ser Phe           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - ACT GCA TAT TTA AGA CGT ATT AAT CGT AAA AC - #A GCG ATA CTG ATA GAA          528                                                                          Thr Ala Tyr Leu Arg Arg Ile Asn Arg Lys Th - #r Ala Ile Leu Ile Glu           #               175                                                           - GCA AGC TGT CAT TTA GGG GCT CTC AGC TCA CA - #G CTT GAT GAA CAA TCT          576                                                                          Ala Ser Cys His Leu Gly Ala Leu Ser Ser Gl - #n Leu Asp Glu Gln Ser           #           190                                                               - ACA TAT CAT ATA AAA CAA TTT GGG CAT TGT AT - #T GGA ATG AGT TAT CAA          624                                                                          Thr Tyr His Ile Lys Gln Phe Gly His Cys Il - #e Gly Met Ser Tyr Gln           #       205                                                                   - ATT ATT GAT GAT ATT CTC GAT TAC ACG AGT GA - #C GAA GCA ACA CTC GGT          672                                                                          Ile Ile Asp Asp Ile Leu Asp Tyr Thr Ser As - #p Glu Ala Thr Leu Gly           #   220                                                                       - AAA CCT GTC GGT AGC GAT ATA AGA AAC GGT CA - #T ATT ACG TAT CCG CTT          720                                                                          Lys Pro Val Gly Ser Asp Ile Arg Asn Gly Hi - #s Ile Thr Tyr Pro Leu           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - ATG GCC GCT ATC GCT AAT TTG AAA GAG CAA GA - #T GAC GAT AAA CTT GAA          768                                                                          Met Ala Ala Ile Ala Asn Leu Lys Glu Gln As - #p Asp Asp Lys Leu Glu           #               255                                                           - GCA GTT GTT AAA CAT TTA ACA TCA ACA TCA GA - #T GAT GAA GTG TAT CAA          816                                                                          Ala Val Val Lys His Leu Thr Ser Thr Ser As - #p Asp Glu Val Tyr Gln           #           270                                                               - TAT ATT GTT TCG CAA GTT AAA CAA TAT GGA AT - #T GAA CCT GCA GAA TTG          864                                                                          Tyr Ile Val Ser Gln Val Lys Gln Tyr Gly Il - #e Glu Pro Ala Glu Leu           #       285                                                                   - CTG AGC AGA AAA TAT GGT GAT AAA GCG AAA TA - #T CAC TTG AGT CAA TTA          912                                                                          Leu Ser Arg Lys Tyr Gly Asp Lys Ala Lys Ty - #r His Leu Ser Gln Leu           #   300                                                                       - CAG GAT AGT AAT ATT AAA GAT TAT TTA GAA GA - #A ATC CAC GAA AAA ATG          960                                                                          Gln Asp Ser Asn Ile Lys Asp Tyr Leu Glu Gl - #u Ile His Glu Lys Met           305                 3 - #10                 3 - #15                 3 -       #20                                                                           # 978              AT TAA                                                     Leu Lys Arg Val Tyr                                                                           325                                                           - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 251 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Gln Asp Ile Tyr Gly Thr Leu Ala Asn Le - #u Asn Thr Lys Leu Lys         #15                                                                           - Gln Lys Leu Ser His Pro Tyr Leu Ala Lys Hi - #s Ile Ser Ala Pro Lys         #             30                                                              - Ile Asp Glu Asp Lys Leu Leu Leu Phe His Al - #a Leu Phe Glu Glu Ala         #         45                                                                  - Asp Ile Lys Asn Asn Asp Arg Glu Asn Tyr Il - #e Val Thr Ala Met Leu         #     60                                                                      - Val Gln Ser Ala Leu Asp Thr His Asp Glu Va - #l Thr Thr Ala Arg Val         # 80                                                                          - Ile Lys Arg Asp Glu Asn Lys Asn Arg Gln Le - #u Thr Val Leu Ala Gly         #                 95                                                          - Asp Tyr Phe Ser Gly Leu Tyr Tyr Ser Leu Le - #u Ser Glu Met Lys Asp         #           110                                                               - Ile Tyr Met Ile Arg Thr Leu Ala Thr Ala Il - #e Lys Glu Ile Asn Glu         #       125                                                                   - His Lys Ile Arg Leu Tyr Asp Arg Ser Phe Ly - #s Asp Glu Asn Asp Phe         #   140                                                                       - Phe Glu Ser Val Gly Ile Val Glu Ser Ala Le - #u Phe His Arg Val Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu His Phe Asn Leu Pro Arg Trp Lys Lys Le - #u Ser Ser Asp Phe Phe         #               175                                                           - Val Phe Lys Arg Leu Met Asn Gly Asn Asp Al - #a Phe Leu Asp Val Ile         #           190                                                               - Gly Ser Phe Ile Gln Leu Gly Lys Thr Lys Gl - #u Glu Ile Leu Glu Asp         #       205                                                                   - Cys Phe Lys Lys Ala Lys Asn Ser Ile Glu Se - #r Leu Leu Pro Leu Asn         #   220                                                                       - Ser Pro Ile Gln Asn Ile Leu Ile Asn Arg Le - #u Lys Thr Ile Ser Gln         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Gln Thr Tyr His Gln Lys Val Glu Glu Gl - #y                             #               250                                                           - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 320 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Lys Leu Lys Ala Met Tyr Ser Phe Leu Se - #r Asp Asp Leu Ala Ala         #15                                                                           - Val Glu Glu Glu Leu Glu Arg Ala Val Gln Se - #r Glu Tyr Gly Pro Leu         #             30                                                              - Gly Glu Ala Ala Leu His Leu Leu Gln Ala Gl - #y Gly Lys Arg Ile Arg         #         45                                                                  - Pro Val Phe Val Leu Leu Ala Ala Arg Phe Gl - #y Gln Tyr Asp Leu Glu         #     60                                                                      - Arg Met Lys His Val Ala Val Ala Leu Glu Le - #u Ile His Met Ala Ser         # 80                                                                          - Leu Val His Asp Asp Val Ile Asp Asp Ala As - #p Leu Arg Arg Gly Arg         #                 95                                                          - Pro Thr Ile Lys Ala Lys Trp Ser Asn Arg Ph - #e Ala Met Tyr Thr Gly         #           110                                                               - Asp Tyr Leu Phe Ala Arg Ser Leu Glu Arg Me - #t Ala Glu Leu Gly Asn         #       125                                                                   - Pro Arg Ala His Gln Val Leu Ala Lys Thr Il - #e Val Glu Val Cys Arg         #   140                                                                       - Gly Glu Ile Glu Gln Ile Lys Asp Lys Tyr Ar - #g Phe Asp Gln Pro Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Thr Tyr Leu Arg Arg Ile Arg Arg Lys Th - #r Ala Leu Leu Ile Ala         #               175                                                           - Ala Ser Cys Gln Leu Gly Ala Leu Ala Ala Gl - #y Ala Pro Glu Pro Ile         #           190                                                               - Val Lys Arg Leu Tyr Trp Phe Gly His Tyr Va - #l Gly Met Ser Phe Gln         #       205                                                                   - Ile Thr Asp Asp Ile Leu Asp Phe Thr Gly Th - #r Glu Glu Gln Leu Gly         #   220                                                                       - Lys Pro Ala Gly Ser Asp Leu Leu Gln Gly As - #n Val Thr Leu Pro Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Tyr Ala Leu Ser Asp Glu Arg Val Lys Al - #a Ala Ile Ala Ala Val         #               255                                                           - Gly Pro Glu Thr Asp Val Ala Glu Met Ala Al - #a Val Ile Ser Ala Ile         #           270                                                               - Lys Arg Thr Asp Ala Ile Glu Arg Ser Tyr Al - #a Leu Ser Asp Arg Tyr         #       285                                                                   - Leu Asp Lys Ala Leu His Leu Leu Asp Gly Le - #u Pro Met Asn Glu Ala         #   300                                                                       - Arg Gly Leu Leu Arg Asp Leu Ala Leu Tyr Il - #e Gly Lys Arg Asp Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Gly Gly Lys Arg Ile Arg Pro Leu                                             #5                                                                            - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ser Leu Ile His Asp Asp                                                     #5                                                                            - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Asp Leu Arg Arg Gly Arg Pro                                                 #5                                                                            - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Leu Ala Gly Asp Gly Leu Leu                                                 #5                                                                            - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Phe Gln Ile Arg Asp Asp Ile Leu Asp                                         #5                                                                            - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Gly Lys Pro Val Gly Ser Asp                                                 #5                                                                            - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24GTCC TTTA                                                  - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               AYGA                                                       - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               GNCC                                                       - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                TCAT C                                                     - (2) INFORMATION FOR SEQ ID NO: 17:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                GCTA A                                                     - (2) INFORMATION FOR SEQ ID NO: 18:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             27   TGWA TYTGRAA                                               - (2) INFORMATION FOR SEQ ID NO: 19:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                TTNC C                                                     - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                TTRT C                                                     - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2451 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - CGAACGTGCT GCACGTAAAG GGCGTAACCC GCAAACTGGT GATGAAATTG AA - #ATCCCAGC         60                                                                          - AAGCAAAGTT CCAGCATTCA AAGCTGGTAA AGCATTAAAA GATGCAGTTA AA - #TAATTGTA        120                                                                          - TCTAAAGCCC ATTATGGGCT TTTTTTATTT GTTCTTATAC CATTTTTTAT AA - #ATTATCGT        180                                                                          - TATAATAATA AAAGGACAAA AATAGAGGTA GATCAATGCG TTATTTACAT AA - #AATTGAAC        240                                                                          - TAGAATTAAA CCGACTTACA AGTCGATATC CATTTTTCAA AAAAATTGCA TT - #TGATGCTG        300                                                                          - AAATCATAAA GCTCGTTGAT GACCTAAATG TCGATGAAAA TGTAAAATGT GC - #GATTGTTG        360                                                                          - CCATTGACAC GAGTATGCGT ATGCAGGATT TTATCAATGA AGATAATAAA GA - #CAGTTTTG        420                                                                          - TACTATCAAC GGATGTTTTG AGTGCTTTAT TTTATAAGTA TTTATCACAG CC - #ATTTTATC        480                                                                          - AGCATGATTT TTTAGTACTG ACGGATTGTG TAAGTCGTAT CAATGAATTA AA - #ATCAATAA        540                                                                          - GAGCAACGAT TACAGACGAA ATTGCTTTGC ATAATATTAA TAAACAAATT CA - #TTATATGT        600                                                                          - TCATACAACC TTATATGAAC AATGAGAAAG TGGTGTCTTA TGAGTAAACA GT - #TAAATGGA        660                                                                          - CAGGAAAAAA GTGAGCTTGT ACATAATGTA TTCCAGAATG TATCGACAAA GT - #ATGACCGC        720                                                                          - CTCAACGATA TCATAAGTTT TAATCAGCAT AAATCCTGGC GTAAATATAC GA - #TGAAACAG        780                                                                          - ATGAATGTTA AAAAAGGGTC GAAAGCACTT GATGTATGCT GCGGTACAGG CG - #ACTGGACA        840                                                                          - ATTCAGATGG CACAGGCTGT CGGTAAAAAT GGTCATGTTA TTGGTCTTGA TT - #TCAGTGAG        900                                                                          - AATATGTTAA GTGTTGCACA AGGAAAAACG AATCATATAC AAAATATTGA AT - #TAATTCAT        960                                                                          - GGTAATGCGA TGGAATTACC ATTTGAAGAT AATATATTTG ATTATACAAC GA - #TTGGTTTT       1020                                                                          - GGTTTACGTA ACTTACCGGA TTATAAAAAA GGATTAGAAG AAATGTATCG TG - #TATTAAAA       1080                                                                          - CCTGGCGGCA TGATTGTTGT TTTAGAAACG AGCCATCCAA CAATGCCAGT AT - #TTAAACAA       1140                                                                          - GGTTACAAAT TATATTTCAA ATACGTTATG CCCCTGTTTG GGAAAGTATT TG - #CTAAGTCT       1200                                                                          - ATGAAGGAAT ATAGCTGGTT ACAGCAAAGT GCTTTTGAAT TTCCTGATAA GT - #ACACGTTA       1260                                                                          - GCACTTTTAA TGGCTGAAAC TGGATTTACA CACATTAAAT TTAAAGGTTT TA - #CTGGTGGC       1320                                                                          - GTGAGTGCGA TGCATCTTGC ATACAAGCCG AAAGAAAAAT AGAATGGATG AT - #TGCTTTGA       1380                                                                          - GTTATAAAGC GTTTTTAAAC CCATATATCA TTGAAGTTGA AAAAAGGTTA TA - #TGAGTGTA       1440                                                                          - TTCAGAGTGA TTCTGAAACG ATAAACAAGG CGGCACACCA TATTTTAAGT TC - #AGGAGGAA       1500                                                                          - AGCGCGTACG TCCGATGTTT GTATTATTAA GTGGTTTTCT GAATGATACA CA - #AAAGGATG       1560                                                                          - ACTTGATTCG TACAGCAGTA TCTCTGGAGC TCGTTCATAT GGCAAGTCTC GT - #TCATGATG       1620                                                                          - ATTACATCGA TAATAGTGAT ATGCGTCGTG GTAATACTTC GGTTCATATA GC - #TTTTGATA       1680                                                                          - AAGACACAGC AATTCGCACA GGACATTTTT TATTAGCACG TGCGTTACAA AA - #TATTGCAA       1740                                                                          - CTATCAATAA TTCGAAATTC CATCAAATTT TTAGTAAAAC GATACTTGAA GT - #TTGTTTTG       1800                                                                          - GTGAATTTGA CCAGATGGCA GATCGATTTA ATTATCCTGT ATCCTTTACT GC - #ATATTTAA       1860                                                                          - GACGTATTAA TCGTAAAACA GCGATACTGA TAGAAGCAAG CTGTCATTTA GG - #GGCTCTCA       1920                                                                          - GCTCACAGCT TGATGAACAA TCTACATATC ATATAAAACA ATTTGGGCAT TG - #TATTGGAA       1980                                                                          - TGAGTTATCA AATTATTGAT GATATTCTCG ATTACACGAG TGACGAAGCA AC - #ACTCGGTA       2040                                                                          - AACCTGTCGG TAGCGATATA AGAAACGGTC ATATTACGTA TCCGCTTATG GC - #CGCTATCG       2100                                                                          - CTAATTTGAA AGAGCAAGAT GACGATAAAC TTGAAGCAGT TGTTAAACAT TT - #AACATCAA       2160                                                                          - CATCAGATGA TGAAGTGTAT CAATATATTG TTTCGCAAGT TAAACAATAT GG - #AATTGAAC       2220                                                                          - CTGCAGAATT GCTGAGCAGA AAATATGGTG ATAAAGCGAA ATATCACTTG AG - #TCAATTAC       2280                                                                          - AGGATAGTAA TATTAAAGAT TATTTAGAAG AAATCCACGA AAAAATGTTA AA - #ACGTGTTT       2340                                                                          - ATTAACAATT GCAAGTAATC CGCTTACAAT GGTAAACTAT TAAGGATTTA TT - #AAATTACA       2400                                                                          #           2451CCATGGA AAAACCACTT TTTATGATTA AACCCTGGAC G                    - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #           30     ACGG AACTTTAGCC                                            - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   TTTA AAATGGCC                                              - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #24:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             28   CGAA ATCGTAAC                                              - (2) INFORMATION FOR SEQ ID NO: 25:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #25:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #               25 TTAC CCTTC                                                 - (2) INFORMATION FOR SEQ ID NO: 26:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #26:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #               25 TTAA AGGCG                                                 - (2) INFORMATION FOR SEQ ID NO: 27:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION: cDNA                                               #27:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #               25 AATA ATCCC                                                 - (2) INFORMATION FOR SEQ ID NO: 28:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 486 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         #28:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - GGTGGCAAGC GCATTAGACC ATTACTTGTT CTGACTACTT TAGATAGTTT AG - #GTGGCAAT         60                                                                          - GCACATGACG GTTTACCATT TGGCATTGCG CTTGAAATGA TTCATACGTA TT - #CTTTAATT        120                                                                          - CACGATGACT TGCCGGCAAT GGATAATGAT GACTATCGTC GCGGTAAACT CA - #CGAATCAT        180                                                                          - AAGCGTTTTG ATGAAGCAAC AGCTATACTC GCTGGAGATG CATTGCTCAC TG - #ATGCTTTT        240                                                                          - CAATGCATTT TAAATACGCA GTTAAACGCA GAAATTAAAT TATCATTGAT TA - #ATTTATTA        300                                                                          - AGTACTGCTT CTGGATCTAA TGGCATGGTT TACGGCCAAA TGCTCGATAT GC - #AAGGTGAA        360                                                                          - CATAAAACAT TGACATTAAA TGAACTGGAA CGTATTCACA TACATAAAAC CG - #GTGANTTG        420                                                                          - ATTCGTGCAG CANTTGTAAG TGCAGGTATC ATANTGANTT TTANTGATGC AC - #AANTGAGC        480                                                                          #          486                                                                - (2) INFORMATION FOR SEQ ID NO: 29:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 741 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION:1..738                                                 #29:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATG AGA AAG TGG TGT CTT ATG AGT AAA CAG TT - #A AAT GGA CAG GAA AAA           48                                                                          Met Arg Lys Trp Cys Leu Met Ser Lys Gln Le - #u Asn Gly Gln Glu Lys           #15                                                                           - AGT GAG CTT GTA CAT AAT GTA TTC CAG AAT GT - #A TCG ACA AAG TAT GAC           96                                                                          Ser Glu Leu Val His Asn Val Phe Gln Asn Va - #l Ser Thr Lys Tyr Asp           #             30                                                              - CGC CTC AAC GAT ATC ATA AGT TTT AAT CAG CA - #T AAA TCC TGG CGT AAA          144                                                                          Arg Leu Asn Asp Ile Ile Ser Phe Asn Gln Hi - #s Lys Ser Trp Arg Lys           #         45                                                                  - TAT ACG ATG AAA CAG ATG AAT GTT AAA AAA GG - #G TCG AAA GCA CTT GAT          192                                                                          Tyr Thr Met Lys Gln Met Asn Val Lys Lys Gl - #y Ser Lys Ala Leu Asp           #     60                                                                      - GTA TGC TGC GGT ACA GGC GAC TGG ACA ATT CA - #G ATG GCA CAG GCT GTC          240                                                                          Val Cys Cys Gly Thr Gly Asp Trp Thr Ile Gl - #n Met Ala Gln Ala Val           # 80                                                                          - GGT AAA AAT GGT CAT GTT ATT GGT CTT GAT TT - #C AGT GAG AAT ATG TTA          288                                                                          Gly Lys Asn Gly His Val Ile Gly Leu Asp Ph - #e Ser Glu Asn Met Leu           #                 95                                                          - AGT GTT GCA CAA GGA AAA ACG AAT CAT ATA CA - #A AAT ATT GAA TTA ATT          336                                                                          Ser Val Ala Gln Gly Lys Thr Asn His Ile Gl - #n Asn Ile Glu Leu Ile           #           110                                                               - CAT GGT AAT GCG ATG GAA TTA CCA TTT GAA GA - #T AAT ATA TTT GAT TAT          384                                                                          His Gly Asn Ala Met Glu Leu Pro Phe Glu As - #p Asn Ile Phe Asp Tyr           #       125                                                                   - ACA ACG ATT GGT TTT GGT TTA CGT AAC TTA CC - #G GAT TAT AAA AAA GGA          432                                                                          Thr Thr Ile Gly Phe Gly Leu Arg Asn Leu Pr - #o Asp Tyr Lys Lys Gly           #   140                                                                       - TTA GAA GAA ATG TAT CGT GTA TTA AAA CCT GG - #C GGC ATG ATT GTT GTT          480                                                                          Leu Glu Glu Met Tyr Arg Val Leu Lys Pro Gl - #y Gly Met Ile Val Val           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - TTA GAA ACG AGC CAT CCA ACA ATG CCA GTA TT - #T AAA CAA GGT TAC AAA          528                                                                          Leu Glu Thr Ser His Pro Thr Met Pro Val Ph - #e Lys Gln Gly Tyr Lys           #               175                                                           - TTA TAT TTC AAA TAC GTT ATG CCC CTG TTT GG - #G AAA GTA TTT GCT AAG          576                                                                          Leu Tyr Phe Lys Tyr Val Met Pro Leu Phe Gl - #y Lys Val Phe Ala Lys           #           190                                                               - TCT ATG AAG GAA TAT AGC TGG TTA CAG CAA AG - #T GCT TTT GAA TTT CCT          624                                                                          Ser Met Lys Glu Tyr Ser Trp Leu Gln Gln Se - #r Ala Phe Glu Phe Pro           #       205                                                                   - GAT AAG TAC ACG TTA GCA CTT TTA ATG GCT GA - #A ACT GGA TTT ACA CAC          672                                                                          Asp Lys Tyr Thr Leu Ala Leu Leu Met Ala Gl - #u Thr Gly Phe Thr His           #   220                                                                       - ATT AAA TTT AAA GGT TTT ACT GGT GGC GTG AG - #T GCG ATG CAT CTT GCA          720                                                                          Ile Lys Phe Lys Gly Phe Thr Gly Gly Val Se - #r Ala Met His Leu Ala           225                 2 - #30                 2 - #35                 2 -       #40                                                                           #                 741 AAA TAG                                                 Tyr Lys Pro Lys Glu Lys                                                                       245                                                           __________________________________________________________________________

What is claimed is:
 1. A method for producing an active thermoresistantprenyl diphosphate synthetase which comprises:separately preparing afirst and second polypeptide subunit of prenyl diphosphate synthetase,the first subunit being derived from a first species of Bacillus and thesecond polypeptide subunit being derived from a second species ofBacillus, the first and second species of Bacillus being different, andmixing the resultant polypeptide subunits to thereby obtain an activethermoresistant enzyme.
 2. The method of claim 1 wherein the firstspecies of Bacillus is Bacillus subtilis and the second species ofBacillus is Bacillus stearothermophilus.
 3. The method of claim 2wherein the polypeptide subunit that is derived from Bacillus subtilishas the amino acid sequence of SEQ ID NO:5, and the polypeptide subunitthat is derived from Bacillus stearothermophilus has the amino acidsequence of SEQ ID NO: 6.